Osteoporosis is the most common metabolic bone disease mainly characterized by low bone mineral density (BMD,--areal BMD (aBMD) unless otherwise specified). Male osteoporosis is a major but most neglected public health problem. Peripheral blood monocytes (PBMs) may act as precursors of osteoclasts, the bone resorption cells, and produce cytokines important for osteoclast differentiation, activation, and apoptosis, and thus represent a major systemic cell for bone metabolism. micro-RNA (miRNAs)-mediated gene expression modifications are important transcriptomic dynamics underlying human diseases, and are involved in osteoclastogenesis in vitro. Next-G RNA- seq has an unparalleled power to comprehensively characterize transcriptome, in particular, revealing novel miRNAs. Therefore, our Hypothesis is: Changes in mRNA and miRNA expression profiles in PBMs underlie male BMD and bone quality/strength variation and can be identified most powerfully by the cutting edge Next-G RNA-seq. Through the Clinical Core, we will recruit and clinically phenotype 200 Caucasian and 100 African American (AA) men, aged 20-30, 150 (100 Caucasians and 50 AA) with high and 150 (100 Caucasians and 50 AA) with low BMD. Bone quality/strength (measured by quantitative CT [QCT] and finite element analyses [FEA]) will also be assessed on each subject. Half of the Caucasians (50 high vs. 50 low BMD subjects) will serve as a ?discovery cohort? and the other half as a ?replication cohort?. These same subjects will all be used in Proj 2 & 3. In Aim 1, we will comprehensively identify mRNAs important to male osteoporosis. We will use the PBMs total RNAs of the ?discovery cohort? to perform RNA-seq-based transcriptome studies to identify mRNAs differentially expressed (DEx) in high vs. low BMD subjects. We will identify the top 10 DEx genes and validate them in the discovery cohort (within-cohort technical validation), the replication cohort (across-cohort biological validation), the AA cohort (across-ethnicity validation), and another independent set of 86 Caucasian female (46 high vs. 40 low BMD) subjects (from our SCOR, for across-sex validation). In silico replication in subjects of different sexes and/or ethnicities will be performed in existing functional genomics datasets. In Aim 2, we will identify/validate DEx miRNAs in high vs. low BMD subjects and the target genes of the top DEx miRNAs. We will identify the top 15 DEx miRNAs, and for each of which, identify top 5 potential target mRNAs through correlation and bioinformatics analyses and also validate their ?targeting? relationship in the above mentioned cohorts/datasets as well as using luciferase-based functional assays. The significant mRNA/miRNA identified above will be tested for their significance for QTC and FEA measures. The data generated in this project will be used for more advanced analyses, such as eQTL/mQTL analyses, gene network analyses, causality analyses and other integrative analyses , e.g., to 1) gain functional insights into the genetic variants and DNA methylation marks identified in Proj 1 & 3; and 2) search for consistent signals of important risk genes through combined analyses of sub-signals at DNA and mRNA levels by data collected in Proj 1 & 2; 3) identify interactions of various epigenetic mechanisms such as miRNA and DNA methylation.